Transgenic Arabidopsis seedlings containing a pathogen-responsive reporter gene allow for convenient high-throughput screening of chemical libraries for compounds that induce plant defense responses. Candidates identified by such screens can be further tested for their ability to protect plants from pathogen-caused diseases. Using Arabidopsis defense signaling mutants, defined regulatory processes that are targeted by a given candidate molecule can be easily narrowed down. Here, we provide a detailed high-throughput screening protocol for library compounds that activate a pathogen-responsive reporter gene in liquid-grown Arabidopsis seedlings.